Bacterial population dynamics after foliar fertilization of almond leaves.

AIMS: To describe the effects of foliar fertilizer application on the bacterial populations of almond tree leaves. METHODS AND RESULTS: We applied a commercially available foliar fertilizer or a water control onto the leaves of almond trees and collected leaves after 1, 7, 14 and 56 days and examined their bacterial populations by 16S rRNA gene sequence analysis. After 1 day, we observed significant differences in 3 of the 4 predominant bacterial phyla, and 5 of the 13 predominant bacterial families. After 7 days, we observed significant differences in all of the predominant phyla, and 8 of the 13 predominant families. After 14 days, the number of significant differences decreased, and after 56 days only 2 of the 13 predominant families differed significantly. CONCLUSIONS: Foliar fertilization significantly altered the bacterial population structure of almond leaves as compared to the water control. While most of the observed perturbation was transient, significant differences remained after 56 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the effects of foliar fertilization on the bacterial populations of almond leaves and provides new insights as to how this process alters the leaf bacterial population structure.

Bacterial populations on the surfaces of organic and conventionally grown almond drupes.

AIMS: To compare the bacterial populations on organically and conventionally grown almond drupes before and after hull split. METHODS AND RESULTS: We constructed 16S rRNA gene libraries, containing approx. 3000 sequences each, from the bacteria from organically and conventionally grown drupes before and after hull split. We observed that before hull split both conventionally and organically grown drupes were colonized by relatively few types of bacteria that were mostly common phyllosphere-associated Proteobacteria. However, the organically grown drupes contained significantly more Alphaproteobacteria and the conventionally grown drupes contained significantly more Gammaproteobacteria. The conventionally grown drupes also contained significantly more sequences associated with the phylum Actinobacteria. After hull split, we observed a significant increase in bacterial diversity, with many newly appearing sequences that were not normally associated with the phyllosphere. CONCLUSIONS: Organic and conventional growing methodologies influence the types of bacteria on almond drupes and hull split results in a burst of microbial diversification. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of organic produce is increasing due to consumer preferences, but it was unknown how this methodology affects the bacterial populations on almond drupes. This is the first study to compare the bacterial populations of organically and conventionally grown almond drupes.

Bacterial population structure and dynamics during the development of almond drupes.

AIMS: To describe the bacterial populations and their dynamics during the development of almond drupes. METHODS AND RESULTS: We examined 16S rRNA gene libraries derived from the bacterial populations on almond drupes at three stages of development: (i) when the drupes were full sized, but before embryo development, (ii) when the drupe hulls first began to split and (iii) when the drupes were fully mature, but before harvesting. Our data revealed that the immature drupes were colonized by relatively few types of bacteria, belonging mostly to common phyllosphere-associated bacteria within the genera Pseudomonas, Pantoea, Methylobacterium and Sphingomonas. However, after the hulls first began to split, the level of bacterial diversity increased and continued to do so until the drupes were fully mature. At the last sampling period, we observed several sequences belonging to bacteria that are not usually associated with the phyllosphere, including some identical to Salmonella enterica. CONCLUSIONS: The bacterial populations on almond drupes before hull split were composed of relatively few types, most of which were commonly associated with the phyllosphere. However, after hull split, the level of microbial diversity increased, which was mostly due to increased levels of bacteria that are not normally associated with the phyllosphere, including Salm. enterica. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the bacterial populations associated with almond drupes and their dynamics during development. Of specific significance is the observation that Salm. enterica was present on the drupes just prior to harvesting, which may represent a critical control point.

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

AIMS: To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. METHODS AND RESULTS: Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. CONCLUSIONS: In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. SIGNIFICANCE AND IMPACT OF THE STUDY: This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Effects of sodium bisulfate on the bacterial population structure of dairy cow waste.

AIMS: To determine the effects of sodium bisulfate (SBS) on the bacterial populations in cattle waste. METHODS AND RESULTS: We applied SBS at 0, 60, 70 or 100 kg week(-1) to cattle waste as it accumulated on the floors of four cattle pens, housing eight cattle each. We observed significant pH decreases in all of the treated wastes on day one; however, the 60 kg week(-1) treatment returned to control levels by day four, while the others remained significantly lower. Heterotrophic plate counts of the waste revealed that all treatments reduced the bacterial populations in the wastes on day one; however, all returned to control levels by day four. The 16S rRNA gene libraries derived from the wastes revealed significant reductions in sequences associated with the phyla Bacteroidetes and Firmicutes and increases in the Proteobacteria, Actinobacteria and Spirochaetes on day one, but resembled the control by day seven. Sequences associated with Escherichia coli increased significantly after SBS application, but became undetectable by day seven. CONCLUSIONS: SBS application significantly alters the bacterial population structure of waste during the first few days of application, but the populations return to almost normal after 7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of SBS to animal waste can reduce emissions; however, biosecurity precautions must be rigorously maintained during the initial application to ensure that pathogenic E. coli is not released into the environment.

Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.

Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of pituitary hormone secretion. Mice with a disrupted D2 dopamine receptor gene had chronic hyperprolactinemia and developed anterior lobe lactotroph hyperplasia without evidence of adenomatous transformation. Unexpectedly, the mutant mice had no hyperplasia of the intermediate lobe melanotrophs. Aged female D2 receptor -/- mice developed uterine adenomyosis in response to prolonged prolactin exposure. These data reveal a critical role of hypothalamic dopamine in controlling pituitary growth and support a multistep mechanism for the induction and perpetuation of lactotroph hyperplasia, involving the lack of dopamine signaling, a low androgen/estrogen ratio, and a final autocrine or paracrine "feed-forward" stimulation of mitogenesis, probably by prolactin itself.

Dopamine as a prolactin (PRL) inhibitor.

Dopamine is a small and relatively simple molecule that fulfills diverse functions. Within the brain, it acts as a classical neurotransmitter whose attenuation or overactivity can result in disorders such as Parkinson's disease and schizophrenia. Major advances in the cloning and characterization of biosynthetic enzymes, transporters, and receptors have increased our knowledge regarding the metabolism, release, reuptake, and mechanism of action of dopamine. Dopamine reaches the pituitary via hypophysial portal blood from several hypothalamic nerve tracts that are regulated by PRL itself, estrogens, and several neuropeptides and neurotransmitters. Dopamine binds to type-2 dopamine receptors that are functionally linked to membrane channels and G proteins and suppresses the high intrinsic secretory activity of the pituitary lactotrophs. In addition to inhibiting PRL release by controlling calcium fluxes, dopamine activates several interacting intracellular signaling pathways and suppresses PRL gene expression and lactotroph proliferation. Thus, PRL homeostasis should be viewed in the context of a fine balance between the action of dopamine as an inhibitor and the many hypothalamic, systemic, and local factors acting as stimulators, none of which has yet emerged as a primary PRL releasing factor. The generation of transgenic animals with overexpressed or mutated genes expanded our understanding of dopamine-PRL interactions and the physiological consequences of their perturbations. PRL release in humans, which differs in many respects from that in laboratory animals, is affected by several drugs used in clinical practice. Hyperprolactinemia is a major neuroendocrine-related cause of reproductive disturbances in both men and women. The treatment of hyperprolactinemia has greatly benefited from the generation of progressively more effective and selective dopaminergic drugs.

Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [seecomments].

The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Distribution and characterization of plasmalemma vesicle protein-1 in rat endocrine glands.

Plasmalemma vesicle protein-1 (PV-1) is an integral membrane protein associated with endothelial cell caveolae and fenestrae. Since endocrine glands are enriched with fenestrated endothelium, we examined the distribution of PV-1 mRNA and protein in endocrine glands and determined its cellular localization. A single transcript was detected by RT-PCR in all endocrine glands examined. A synthetic peptide was used to generate antibodies for Western blotting and immunohistochemistry (IHC). Western blotting of membrane fractions from lung, pituitary, adrenal, testis and PV-1-transfected Cos-1 cells revealed a major 65 kDa protein. This protein binds to heparin with high affinity. Using IHC, PV-1 was localized to both endothelial cells of the adrenal zona reticularis and chromaffin cells of the medulla. In the pancreas, PV-1 expression was restricted to a few cells in the islets of Langerhans that partially overlap with somatostatin-positive delta-cells. In both neonatal and adult pituitaries, strong PV-1 immunoreactivity was detected in neural lobe pituicytes in a pattern similar to that of glial fibrillary acidic protein (GFAP). PV-1 and GFAP expression was seen in the adult, but not neonatal, intermediate lobe. Endothelial cells throughout the neonatal anterior lobe were PV-1 positive, but PV-1 in the adult was restricted to some endothelial and endocrine cells localized near the margins of lobe. In the adult testis, strong PV-1 expression was seen in germ cells within the seminiferous tubules that varied with the stage of spermatogenesis. In contrast, PV-1 in the neonatal testis was localized to the interstitial cells but not seminiferous tubules. In the ovary, PV-1 was expressed in stromal endothelial cells as well as the thecal layer of developing follicles. Over half the corpus luteal cells were positive for PV-1. Our data have shown that PV-1 is not restricted to endothelial cells but is localized in many types of endocrine and non-endocrine cells. Furthermore, PV-1 expression in the pituitary and testis is developmentally regulated.

Functional mapping of neural sites mediating prolactin-induced hyperphagia in doves.

Microinjections of prolactin (PRL) into the ventromedial nucleus of the hypothalamus (VMN) or the preoptic area (POA) have been previously shown to increase food intake and body weight in ring doves. In an attempt to corroborate these results and to provide a more complete map of PRL-sensitive brain sites mediating the orexigenic action of PRL, a microinjection procedure was employed in the present study that delivered PRL or saline vehicle in extremely small volumes (10 nl/injection) to a variety of diencephalic sites in dove brain that had been previously demonstrated to contain high concentrations of PRL receptors. Estimates obtained from one female subject given a single 10 nl injection of [125I]ovine PRL into the VMN supported the claim that such injection volumes resulted in limited diffusion, as 80% of the tissue radioactivity was found within a 280 mm area surrounding the injection site at 30 min after injection. Food intake of cannulated male doves in the mapping study was monitored daily during a 6 day baseline period, an initial 4 day treatment period, a 6-12 day post-treatment recovery period, and a second 4 day treatment period. Approximately half of the birds received PRL injections (50 ng/10 nl twice daily) and saine vehicle injections (10 nl twice daily) during the first and second treatment periods, respectively, while remaining birds received these treatments in the reverse order. No significant changes in food intake across baseline, vehicle, post-treatment, or PRL treatment periods were observed in birds with injection sites in the lateral POA, paraventricular nucleus of the hypothalamus (PVN), or the medial-basal hypothalamic region between the tuberal hypothalamus (TU) and VMN. In contrast, injections of PRL into the VMN area, medial POA, or TU resulted in average daily food intake values that significantly exceeded those recorded during other periods. The most robust feeding response was seen in the VMN group, where PRL injections resulted in a 58% increase in food intake over that recorded during injection of vehicle. This increase was significantly greater than that observed following PRL injections into the mPOA (26%) or the TU (32%). These findings suggest that the VMN may be a primary site of PRL action in promoting hyperphagia in this species, although PRL effects at other diencephalic loci, such as the mPOA and TU, may also contribute to the orexigenic action of this hormone.

Role of the ventromedial hypothalamus in prolactin-induced hyperphagia in ring doves.

Prolactin (PRL) strongly stimulates feeding activity and body weight gain in ring doves, and of the brain loci tested to date, the ventromedial hypothalamus (VMH) is the most effective site of PRL action in promoting these changes. To determine if the VMH is essential for this response, we examined the effects of VMN destruction on spontaneous feeding and on changes in food intake induced by intracerebroventricular (i.c.v.) injections of PRL. Male birds were selectively destroyed by radiofrequency lesions (n = 6). A group of sham-lesioned males (n = 6) served as controls. Lesioned birds exhibited a transient increase in food intake that peaked around the seventh postoperative day and declined to baseline levels by day 12. In contrast to this pattern, body weights of lesioned birds increased in parallel with food intake, but remained elevated throughout the 3-week postoperative period. During the peak period of hyperphagia in the lesioned group, food intake and body weight increases were two to three times greater in lesioned birds than in controls. After postoperative feed intake had stabilized, each bird received 5 daily i.c.v. injections of ovine PRL. Food intake and body weight increased dramatically in both groups in response to PRL treatment, and no group differences were observed in response to magnitude. We conclude that VMH destruction strongly perturbs feeding and body weight regulation in doves. However, VMH integrity is not essential for the expression of PRL-induced hyperphagia.

Changes in bioactive prolactin-like activity in plasma and its relationship to incubation behavior in breeding ring doves.

Previous radioimmunoassay (RIA) data indicate that plasma prolactin (PRL) is elevated during the late incubation and the early posthatching periods of the ring dove breeding cycle. Although these changes are temporally associated with changes in PRL-dependent crop sac growth, the precise relationship between immunoreactive and bioactive PRL has not been directly examined. To investigate this question and to further explore the relationship between sitting behavior and PRL secretion, we used rat Nb2 lymphoma cell proliferation to estimate the concentration of bioactive PRL-like activity (PLA) in the plasma of breeding ring doves. Serial dilutions of dove pituitary homogenate and dove plasma stimulated mitogenic responses that were parallel to those observed with purified ovine PRL. Changes in plasma PLA during the breeding cycle closely resembled changes in PRL that have been previously reported by RIA, although the relative changes in PLA were more pronounced. In both sexes, PLA remained at basal levels prior to egg laying and during early incubation (Day 4-5) but then abruptly increased to reach peak values near the time of hatching (Day 14-15). Activity remained high for 3-4 days after hatching, declined gradually thereafter, and returned to baseline values by Posthatching Days 14-17. Plasma PLA levels of birds sampled at the end of incubation were correlated with those of their breeding partners. In the majority of pairs, females had higher PLA levels than their mates at this stage even though no significant overall sex differences in PLA levels were observed. Plasma PLA declined precipitously in birds that were nest deprived on the last day of the incubation period. Nevertheless, plasma PLA levels of normally breeding birds at the end of incubation were not correlated with the average time spent in the nest during the incubation period. However, day-to-day variability in time spent in the nest correlated negatively with plasma PLA in incubating males, and females exhibited a similar trend that approached significance. These data suggest (1) that published RIA estimates of PRL are reasonably accurate reflections of changes in bioactive PLA in dove plasma and (2) that while sitting duration itself is not strongly related to plasma PLA, large day-to-day fluctuations in nest occupation time are associated with reduced PLA levels in incubating doves.